Targeted brain delivery of RVG29‐modified rifampicin‐loaded nanoparticles for Alzheimer's disease treatment and diagnosis

Abstract Alzheimer's disease (AD) is an aging‐related neurodegenerative disease. The main pathological features of AD are β‐amyloid protein (Aβ) deposition and tau protein hyperphosphorylation. Currently, there are no effective drugs for the etiological treatment of AD. Rifampicin (RIF) is a semi‐synthetic broad‐spectrum antibiotic with anti‐β‐amyloid deposition, anti‐inflammatory, anti‐apoptosis, and neuroprotective effects, but its application in AD treatment has been limited for its strong hydrophobicity, high toxicity, short half‐life, low bioavailability, and blood–brain barrier hindrance. We designed a novel brain‐targeted and MRI‐characteristic nanomedicine via loading rabies virus protein 29 (RVG29), rifampicin, and Gd on poly (l‐lactide) nanoparticles (RIF@PLA‐PEG‐Gd/Mal‐RVG29). The cytotoxicity assay demonstrated that RIF@PLA‐PEG‐Gd/Mal‐RVG29 had favorable biocompatibility and security. Fluorescence imaging in vivo showed that PLA‐PEG‐Gd/Mal‐RVG29 could deliver rifampicin into the brain by enhancing cellular uptake and brain targeting performance, leading to improvement of the bioavailability of rifampicin. In in vivo study, RIF@PLA‐PEG‐Gd/Mal‐RVG29 improved the spatial learning and memory capability of APP/PS1 mice in the Morris water maze, as compared to rifampicin. Immunofluorescence, TEM, immunoblotting, and H&E staining revealed that RIF@PLA‐PEG‐Gd/Mal‐RVG29 reduced Aβ deposition in hippocampal and cortex of APP/PS1 mice, improved the damage of synaptic ultrastructure, increased the expression level of PSD95 and SYP, as well as reduced the necrosis of neurons. These findings suggest that RIF@PLA‐PEG‐Gd/Mal‐RVG29 may be an effective strategy for the treatment of AD.

lation in AD mice. [13][14][15][16] Although RIF has definite potential efficacy on anti-dementia, anti-inflammatory, anti-apoptosis, and neuroprotection, it occasionally causes severe side effects (such as liver damage). In addition, oral RIF has a very low utilization efficiency after intestinal absorption and first-pass effects on the liver. Therefore, it is necessary to find a novel targeting drug delivery approach to avoid the first-pass effect on the liver, and the side effects, that will achieve better therapeutic effects.
Although drug therapy has become an effective strategy in the treatment of AD and related neurodegenerative diseases. However, only a few drugs are available for the clinical treatment of AD. These traditional drugs may cause higher side effects on normal cells, and their therapeutic effect is still limited. Molecular chaperones were found to be effective inhibitors of neurodegenerative diseases due to their ability to inhibit intermolecular interactions between abnormal proteins. 17 Recently, Huang et al. prepared a novel mixed-shell polymeric micelles (MSPMs) artificial chaperone to efficiently inhibit the accumulation of Aβ and reduce neuronal cell cytotoxicity. 18 The presence of the blood-brain barrier (BBB) is a major obstacle to treating neurodegenerative diseases, which prevents the access of nano-drugs to the brain from the blood. In recent years, functional nanomaterials have been used in the treatment of neurodegenerative diseases due to their excellent biocompatibility and drug delivery properties. Encapsulating small drug molecules in nanomaterials can improve their delivery efficiency in the central nervous system. 19,20 Some targeted ligands, such as insulin and lactoferrin have been employed to carry therapeutic drugs into the brain. 21,22 Brain-targeted polymer nanoparticles that can deliver drugs through the BBB are considered a promising strategy for the treatment of neurological diseases. 23 These polymer nanoparticle drug delivery systems can carriage therapeutic drug molecules that would otherwise be impermeable to the BBB. In recent years, brain-targeted drug delivery systems have been considered as potential therapeutics for AD. 24,25 However, the brain-targeting properties and drug delivery efficiency of existing polymer drug delivery systems are still limited.
Brain-targeted RVG29, a 29 amino acid peptide originating from the rabies virus glycoprotein (RVG), has a specific binding effect on the nicotinic acetylcholine receptors (nAchR) on neuronal cells. 26 Because nAchR is widely present on the surface of neurons and capillary endothelial cells in the brain, RVG29 can effectively cross the BBB into the brain via nAchR-mediated endocytosis. 27 It has been demonstrated that biodegradable polyethylenimine modified with RVG peptide as targeting ligands for neuronal cells can promote gene delivery to the brain. 28 Kumar et al. found that a chimeric peptide combined with nona(D-arginine) peptide and RVG could bind and deliver siRNAs to the central nervous system, leading to objective gene silencing in the brain. 29 So far, RVG29-modified nano-drug delivery systems, including nucleic acids, 30 biologically derived or synthetic nanoparticles, [31][32][33] nano-device 27,34 and liposomes, 35 can transport a variety of volume-large drugs across the BBB and promote drug accumulation in the brain when administered systemically in the treatment of neurodegenerative diseases. Targeted drug delivery systems can not only enhance the efficiency of drug treatment of lesions, but also help reduce the side effects of drugs on normal cells. Therefore, RVGmodified drug carriers provide a safe and non-invasive potential therapeutic strategy for drug delivery across the BBB to treat neurological diseases.
Herein, we designed and constructed nanoparticles by assembling and synthesizing biodegradable polylactic acid, contrast-enhanced gadolinium (Gd), and inflammation-eliminated rifampicin. Then its surface was modified by targeting protein RVG-29 to obtain a novel brain-targeted nanomedicine RIF@PLA-PEG-Gd/Mal-RVG29. This nanomedicine can penetrate the BBB of mice with AD disease to achieve targeted therapy, which is beneficial to alleviate brain inflammation, delay the pathological process of AD and improve the cognitive function of mice. In addition, the magnetic resonance imaging characteristics of Gd have been used to diagnose AD in vivo and evaluate the efficacy of nanomedicine, providing new strategies and methods for accurate diagnosis and treatment of AD. Currently, the potential application of RVG29-modified delivery system combined brain-targeted therapy, anti-inflammatory, and magnetic resonance imaging functions for treatment and diagnosis of AD remains unexplored.

| Materials
Poly(L-lactide)-poly(ethylene glycol)-maleimide (PLA-PEG-Mal, MW of PLA is 10 k and MW of PEG is 5k, >90%) and poly(L-lactide)-poly (ethylene glycol)-amine (PLA-PEG-NH 2 , MW of PLA is 10k and MW of PEG is 5k, >90%) were purchased from Xi'an Ruixi Biotechnology    The mice in each group were equally divided into female and male mice. Both WT mice and APP/PS1 mice were injected with the same volume of PBS. All mice were administered via tail vein injection with once every 3 days and a total of eight times. In this test, a total of 32 Tg mice aged 9-10 months were used. Eight WT mice from the same batch were used as the control group. These mice were ran-

| Tissue preparation
Morris's water maze (MWM) experiment was described in the Supporting information. After the MWM experiment, the mice were deeply anesthetized with tribromoethanol, and cardiac perfusion was performed with ice-cold saline and 4% paraformaldehyde until the effluent became clear and transparent. Then the brain was quickly removed and fixed in 4% paraformaldehyde overnight. The brain was embedded in paraffin for H&E staining and immunofluorescence experiments. To analyze the changes in the synaptic structure of hippocampal CA1 region through transmission electron microscopy, cardiac perfusion was performed with saline until the effluent was clear and transparent. Subsequently, it was fixed by perfusion with 2.5% glutaraldehyde. The CA1 region of each hippocampus was quickly separated and then stored in 2.5% glutaraldehyde at 4 C for further use. For the western blot test, after the mice were bled from the eyeballs, fresh brain tissue was quickly removed on the ice platform, and then stored at À80 C for further use.

| Hematoxylin-eosin staining
Hematoxylin-eosin (H&E) staining was used to observe the morphological changes of hippocampal CA1 neurons in mice. The paraffinembedded brain tissue was cut into 4 μm sections. These brain tissue sections were dewaxed with xylene and hydrated with ethanol. Then nucleus was stained with hematoxylin, and the cytoplasm was stained with eosin. Tissue sections were sealed and stored between slides and coverslips after dehydration by using ethanol. The pathological changes of neurons in the hippocampal CA1 area were observed under the light microscope.

| Immunofluorescence staining
The paraffin-embedded brain tissue was cut into 4 μm sections. After preheating, these brain tissue sections were then dewaxed in xylene.
Then, these brain tissue sections were fixed in ethanol with different concentration gradients (100%, 90%, 80%, 70%, 60%), followed by antigen retrieval. After blocking with 5% bovine serum albumin for 1 h, the sections were incubated with Aβ antibody (1:100) overnight at 4 C. The next day, these sections were washed with PBS and then incubated with the TRITC donkey anti-rabbit secondary antibody (1:500, Abcam) at room temperature for 1 h. Finally, it was stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI). An inverted fluorescence microscope was used to observe the Aβ deposition in the cerebral cortex and hippocampus. The data were analyzed by Image-pro Plus 6.0.

| Western blot analysis
The western blot antibodies used were as follows:

| Statistical analysis
The data were first tested for equal variance and normality, and then showed as mean ± SD. Comparisons among multiple groups were made using one-way ANOVA followed by Bonferroni post hoc pairwise comparisons. Differences were deemed statistically significant where p < 0.05. Statistical analysis of the data was performed by using SPSS19.0 statistical software. and encapsulation efficiency was 49.2%. The loading capacities of most nanomaterials were less than 10% in other researches. 36,37 Notably, our study discovered a new material that increases the loading capacity to 16.7% and 16.4%. As shown in Figure  studies. 38 Most transport across the BBB occurs in a selective and tightly regulated manner and is controlled by carrier-mediated transport and receptor-mediated transport (RMT). 39    nanoparticle distribution in the brain tissue should also be confirmed from the brain tissue section using Cy5.5-labeled nanoparticles in this study.

| MRI of amyloid plaque detection in mice
Gadolinium (Gd) chelates are the most widely used MRI contrast agents. Gd can be used as an MRI contrast agent to detect amyloid plaques. By adjusting the magnetic resonance signal, Gd is able to shorten the T1 relaxation time of surrounding protons and increase the contrast between amyloid plaques and brain parenchyma. 44 The hydrophilic Gd contrast agent can increase the signal of the tissue around the amyloid plaque and make the amyloid plaque appear black spots. 45,46 In order to confirm the diagnostic properties of the nanoparticles for AD, MRI with Gd-assisted imaging was performed on 10-month-old WT and APP/PS1 mice, respectively. Immunofluorescence staining showed that amyloid protein was not detected in WT mice (Figure 5c), but was detected in the hippocampus and cortex of APP/PS1 mice. Before contrast agent injection, hypointense spots were not visible in the MRI of WT and APP/PS1 mice (Figure 5a,d). After contrast agent injection, the hypointense spots could be seen in the cortex and hippocampus of APP/PS1 mice (Figure 5e), but they did not show in WT mice (Figure 5b). Immunofluorescence staining of Aβ in brain tissue further confirmed that the hypointense spots on magnetic resonance images were amyloid plaques (Figure 5f). The above results support that nanoparticles have the ability to diagnose amyloid plaques in vivo. Therefore, the prepared nanomedicine has the potential in the diagnosis of AD and evaluation of drug efficacy through detecting amyloid plaques in the brain by MRI. Distinct from the reported materials, such as the PAMAM-PEG-RVG29/ DNA, 31 we designed and fabricated the RIF@PLA-PEG-Gd/Mal-RVG29, which offers the advantages of biocompatibility, brain targeting, the controllable release of carried drugs, and the ability of disease diagnosis. However, the toxicity of NPs needs to be concerned. Gd is a toxic MRI agent. The leaking of Gd from the NPs will be studied in our future studies. Three important areas of spatial memory function and the transition from short-term memory to long-term memory are the hippocampus, entorhinal cortex, and cingulate cortex. The damage to these areas is related to the memory loss of AD. 48 The pathogenesis of AD is complex. The accumulation and deposition of Aβ play a key role in the pathogenesis of AD. 49 The formation and deposition of Aβ in the cortex and hippocampus are directly related to learning and memory deficits in AD. 50 Previous studies reported that rifampicin has the ability to enhance Aβ clearance. 13 As shown in Figure 7, we used immunoflu- ). This result indicated that RVG29-modified nanoparticles loaded with rifampicin was a promising approach to reducing Aβ deposition. In our study, we showed the levels of Aβ in the brain of these mice by Immunofluorescence staining, but ELISA or Western blot will be included for quantitative comparison in our future studies.

| Effect of RIF@PLA-PEG-Gd/Mal-RVG29 on synaptic structure and synapse protein
Synaptic plasticity is the basis of learning and memory. 51 The deposition of Aβ leads to the destruction of synaptic signaling pathways and dendritic spines, thereby affecting the morphology and function of synapses and leading to memory and behavior defects. 52 The production of Aβ at the dendrites and axons will partly reduce the number and plasticity of synapses. 53 Postsynaptic density protein 95 (PSD95) and synaptophysin (SYP) is closely related to synaptic function proteins. PSD95 plays an important role in the plasticity, stability of synapses, and the repair of peripheral nerves after injury. 54 As a presynaptic plasticity-related protein, SYP expression indirectly reflects the number, distribution, and density of synapses. 55 Therefore, the effect of RIF@PLA-PEG-Gd/Mal-RVG29 on synaptic ultrastructure and synaptic protein PSD95 and SYP in Tg mice was studied. As shown in Figure 8, RIF@PLA-PEG-Gd/Mal-RVG29 could prevent Aβ(1-40)-induced neurotoxicity on rat pheochromocytoma PC12 cells. 57 Rifampicin pretreatment can protect PC12 cells from rotenone-induced cell death. Rifampicin significantly inhibited rotenone-induced apoptosis by reducing oxidative stress in mitochondria. 58 An in vivo study showed that rifampicin attenuates MPTPinduced neurodegeneration in nigrostriatal dopamine neurons of mouse brains. 59 The above research results show that rifampicin has a neuroprotective effect in vitro and in vivo in mice. In this study, H&E staining was performed to evaluate the effect of RIF@PLA-PEG-Gd/ Mal-RVG29 on neurons in the brain of Tg mice (Figure 9). For WT + PBS group, the neuronal cells were neatly arranged, and the cell morphology was normal and uniformly distributed (Figure 9a

| In vivo toxicity studies of nanoparticles
Automatic biochemical analyzer was used to evaluate the toxicity of RIF@PLA-PEG-Gd/Mal and RIF@PLA-PEG-Gd/Mal-RVG29 in each.
We detected the creatinine value of mice in each group. There was no significant difference among groups in the creatinine value of mice ( Figure S3). This result indicated that the biosafety of NPs is reliable in vivo.

| CONCLUSION
In this work, we successfully designed and prepared rifampicin-loaded brain-targeted nanoparticles (RIF@PLA-PEG-Gd/Mal-RVG29). The physical and chemical properties of these nanoparticles were revealed by investigating the drug loading, encapsulation efficiency, particle size, in vitro drug release, cytotoxicity, and distribution in the brain.
Effects of RIF@PLA-PEG-Gd/Mal-RVG29 on the improvement of cognitive function, Aβ deposition, synaptic ultrastructure, a synaptic protein, and changes in neuron morphology in APP/PS1 mice with AD were further explored. The prepared RIF@PLA-PEG-Gd/Mal-RVG29 can improve the bioavailability of rifampicin due to its good biocompatibility, brain targeting, and controlled release drug. In vivo, our study showed that these nanoparticles can effectively improve cognitive impairment, reduced Aβ deposition and neuronal death, and promoted synaptic remodeling in AD mice. In addition, the prepared nanomedicine has the potential in diagnosis of AD and evaluation of drug efficacy through MRI. Thus, RIF@PLA-PEG-Gd/Mal-RVG29 is a promising biodegradable material for AD treatment.

CONFLICT OF INTEREST
There are no conflicts to declare.

DATA AVAILABILITY STATEMENT
Data available on request due to privacy/ethical restrictions.